IL-15 induces IFN-γ production by B cells. (A,B) Total B cells (B220⁺) from Ii^−/− mice (immature) were suspended in 5 mL RPMI plus 10% FCS and incubated in the presence or absence of IL-15 (30 ng/mL) for various time periods as indicated. RT-PCR using primers for IFN-γ and HPRT was performed as described in “RNA isolation and reverse transcription.” (C,D) Total B cells (B220⁺) from Ii^−/− mice (immature) were suspended in 5 mL RPMI plus 10% FCS and incubated in the presence or absence of IL-15 (30 ng/mL) for various time periods as indicated. Quantitative real-time PCR was performed using primers for IFN-γ and HPRT as described in “Quantitative real-time RT-PCR.” Results shown are representative of 3 separate experiments. (E,F) Total lymphocytes from Ii^−/− mice (E) or control mice (F) were incubated in the presence or absence of IL-15 (30 ng/mL) overnight. The cells were stained for B220, and IFN-γ protein levels were analyzed by intracellular staining as described in “Intracellular staining.” The graphs show the percentage of B220⁺ and IFN-γ⁺ B cells minus the background of cells stained with an isotype control antibody. (G) Total B cells from control (mature) and Ii^−/− (immature) mice were suspended in 5 mL RPMI plus 10% FCS and incubated in the presence or absence of IL-15 (30 ng/mL) for 1 hour. RNA was isolated and subjected to RT-PCR was preformed using primers for IFN-γ and HPRT as described in “RNA isolation and reverse transcription.” (H) Total lymphocytes from control and Ii^−/− mice were incubated overnight in the presence or absence of IL-15 (30 ng/mL) and stained for B220, and IFN-γ levels were analyzed by intracellular staining. The graph shows the fold increase in IFN-γ protein levels following treatment in the 2 populations. The results are representative of 3 separate experiments. Error bars represent SD.